The four subclasses of human IgG behave differently in a number of respects, such as propensity to dimerize, susceptibility to fragmentation by plasmin, and ability to activate complement. In addition, antibody activity toward some antigens has been reported to be subclass restricted. In order to further investigate these differences, it is necessary to separate and purify the individual IgG subclasses from polyclonal human IgG. Several methods for achieving this separation have been examined, including ph-gradient chromatography on Protein A-Sepharose and QAE-Sepharose and a variety of salt-gradient chromatographic procedures with SP-Sepharose and DEAE-Sepharose. Although some of these methods gave substantial enrichment of particular subclasses, none allowed attainment of the desired purity (greater than 95%) of all four subclasses. For this reason, it was felt that the ultimate purification of IgG subclasses will require an immunoaffinity method. To that end, mouse monoclonal antibodies to all four subclasses have been purchased, and these will be used to prepare immunoaffinity columns for final purification of the IgG subclasses from pooled IgG.